Lipase-containing polymeric coatings for the facilitated removal of fingerprints

ABSTRACT

A substrate or coating is provided that includes a lipase with enzymatic activity toward a component of a fingerprint. Also provided is a process for facilitating the removal of fingerprints is provided wherein an inventive substrate or coating including a lipase is capable of enzymatically degrading of one or more components of the fingerprint to facilitate fingerprint removal from the substrate or said coating. Applying heat to the substrate or coating increases the rate of fingerprint removal.

FIELD OF THE INVENTION

The present invention relates generally to coating compositionsincluding bioactive substances and methods of their use to facilitateremoval of fingerprints. In specific embodiments, the invention relatesto methods for fingerprint removal by incorporating lipase into or onpolymer composite materials to degrade fingerprint components.

BACKGROUND OF THE INVENTION

Many consumer products such as cell phones, touch-screen displays,automobile door handles, etc., are subject to frequent contact withhands and fingers. As a result, the residue of fingerprints often leavesunpleasant marks on the surface deteriorating the cosmetic appearance ofthe products.

Prior art approaches aim to reduce the deposition of the fingerprintstains on a surface and facilitate its removal capitalize on the“lotus-effect” where hydrophobic, oleophobic and super-amphiphobicproperties are conferred to the surface by polymeric coatings containingappropriate nanocomposites. An exemplary coating contains fluorine andsilicon nanocomposites with good roll off properties and very high waterand oil contact angles. When used on rough surfaces like sandblastedglass, nanocoatings may act as a filler to provide a fingerprintresistance. A drawback of these “passive” technologies is that theyrequire water-rinsing to finally remove the fingerprints from thesurface. In addition, these materials are not suitable for use in highgloss surfaces because the lotus-effect is based on surface roughness.

The photocatalyst Ti0₂ was used to promote active fingerprintdecomposition of fingerprint stains in U.S. Pat. Appl. Publ.2009/104086. A major drawback to this technology is its limitation touse on inorganic surfaces due to the oxidative impairment of the polymercoating by Ti0₂.

Therefore, there is a need for new materials or coatings that canactively promote the removal of fingerprints on organic surfaces or inorganic coatings and minimize the requirement for maintenance cleaning.

SUMMARY OF THE INVENTION

A composition and method for fingerprint removal from a substratesurface is provided. The method includes associating a lipase with asubstrate or a coating such that the lipase is capable of enzymaticallydegrading a component of a fingerprint.

A method optionally includes heating the substrate or applying heat tothe surface of the substrate. In some embodiments heating is at least 5degrees Celsius above ambient temperature. The substrate or surfacethereon is optionally heated to between 40 and 125 degrees Celsius.Heating is optionally continued for at least 30 minutes, illustrativelyfor between 30 minutes to 6 hours.

The composition includes a substrate or coating containing a lipase. Thecomposition optionally includes an organic crosslinkable ornon-crosslinkable polymer resin. The resin optionally has a functionalgroup of acetoacetate, acid, amine, carboxyl, epoxy, hydroxyl,isocyanate, silane, vinyl, or combinations thereof. Specific examples ofresins include aminoplasts, melamine formaldehydes, carbamates,polyurethanes, polyacrylates, epoxies, polycarbonates, alkyds, vinyls,polyamides, polyolefins, phenolic resins, polyesters, polysiloxanes, orcombinations thereof. In particular, a resin is optionally ahydroxyl-functionalized acrylate resin.

A substrate or coating has one or more associated lipase enzymes. Alipase is optionally lipoprotein lipase, acyl glycerol lipase,hormone-sensitive lipase, phospholipase A1, phospholipase A2,phospholipase C, phospholipase D, phosphoinositide phospholipase C, alysophospholipase, or a galactolipase. In particular embodiments, alipase is a triacylglycerol lipase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents fingerprint removal on a substrate coated with alipase containing coating;

FIG. 2 represents increased fingerprint removal rates on a plate coatedwith a lipase containing coating;

FIG. 3 represents similar removal rates of fingerprints from multiplesources.

FIG. 4 represents a schematic of an inventive method.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following description of embodiment(s) of the invention is merelyexemplary in nature and is in no way intended to limit the scope of theinvention, its application, or uses, which may, of course, vary. Theinvention is described with relation to the non-limiting definitions andterminology included herein. These definitions and terminology are notdesigned to function as a limitation on the scope or practice of theinvention but are presented for illustrative and descriptive purposesonly.

The present invention is based on the catalytic activity of a lipaseenzyme to selectively degrade and volatilize components of fingerprints,thus, promoting active fingerprint removal. Fingerprint stains typicallyinclude components of sweat gland secretion and sebum which includeslipids, wax, and cellular debris. Several of the substances of sebum arelipophilic and have low volatility such as squalene and wax esters.

The lipase that is either immobilized in coatings or substratescatalyzes the hydrolysis, esterification, or transesterification oflipids including triacylglycerols, cholesterol esters, and otherfingerprint components into smaller molecules. The smaller molecules mayhave higher volatility than their precursors and more easily vaporize atambient or elevated temperatures thereby allowing for complete stainremoval. Without being limited to one particular theory, it is believedthat the resulting degradation products may have lower boiling points orreduced adhesion promoting increased vaporization either upon heating orincubation at ambient temperatures. Thus, the invention has utility as acomposition and method for the active removal of fingerprints fromsurfaces.

The inventive methods and compositions are generally referred to hereinas a lipase associated with a substrate for exemplary purposes only. Oneof ordinary skill in the art appreciates that the description is equallyapplicable to coatings either on a substrate or prior to application toa substrate.

An inventive method includes providing a substrate or a coating with alipase or analogue thereof such that the lipase or analogue thereof isenzymatically active and capable of degrading one or more components ofa fingerprint. In particular embodiments, a fingerprint is based onbioorganic matter such as that derived from the skin of a subject.

A fingerprint as defined herein is a bioorganic stain, mark, or residueleft behind after an organism touches a substrate or coating. Afingerprint is not limited to marks or residue left behind after asubstrate is touched by a finger. Other sources of bioorganic stains areillustratively, palms, toes, feet, face, any other skin surface area,hair, stains from fats used in cooking such as cis-fatty acids, or fattyacids from any other source.

A lipase is optionally a lipoprotein lipase, acylglycerol lipase such astriacylglycerol lipase, hormone-sensitive lipase, phospholipase A1,phospholipase A2, phospholipase C, phospholipase D, phosphoinositidephospholipase C, a lysophospholipase, a galactolipase, or combinationsor analogues thereof. An analogue of a lipase is optionally a fragmentof a lipase. An analogue of a lipase is a polypeptide that has somelevel of activity toward a natural or synthetic substrate of a lipase.An analogue optionally has between 0.1% and 200% the activity of awild-type lipase.

Specific examples of lipase include Lipase AP4, Lipase AP6, Lipase AP12,Lipase M-AP5, Lipase M-AP10 and Lipase M-AP20 (manufactured by AmanoPharmaceutical Co7., Ltd.), Lipase Saiken (manufactured by Osaka SaikinKenkyusho), Lipase MY (manufactured by Meito Sangyo), or Lipase B(Candida antarctica or Candida rugosa). It is also possible to usemultiple enzyme preparations having lipase activity. For example, mixeddigestive enzyme preparations are operable such as Biodiastase,Biodiastase 500, Biodiastase 700, Biodiastase 1000, Biodiastase 2000,Pancreatin, Pancreatic Digestive Enzyme TA and Pancreatic DigestiveEnzyme 8AP (manufactured by Amano Pharmaceutical Co., Ltd.),Biotamylase, Biotamyolase S, Biotalase A-1000, Biotalase P-1000 andDenapsin 10 (manufactured by Nagase Seikagaku Kogyo), Cellulosin AP andProlicin (manufactured by Ueda Kagaku), Takadiastase (manufactured bySankyo), Sumizyme (manufactured by Shin Nippon Kagaku Kogyo) andBiotamylase (manufactured by Nagase Sangyo).

A lipase is optionally derived from Acinetobacter, Aedes aegypti,Anguilla japonica, Antrodia cinnamomea, Arabidopsis rosette, Arabidopsisthaliana, Arxula adeninivorans, Aspergillus niger, Aspergillus oryzae,Aspergillus tamarii, Aureobasidium pullulans, Avena sativa, Bacilluslicheniformis, Bacillus sphaericus, Bacillus stearothermophilus,Bacillus subtilis, Bacillus thermocatenulatus, Bacillus thermoleovorans,Bombyx mandarina, Bombyx mori, Bos Taurus, Brassica napus, Brassicarapa, Burkholderia cepacia, Caenorhabditis elegans, Candida albicans,Candida antarctica, Candida deformans, Candida parapsilosis, Candidarugosa, Candida thermophila, Canis domesticus, Chenopodium rubrum,Clostridium beijerinckii, Clostridium botulinum, Clostridium novyi,Danio rerio, Galactomyces geotrichum, Gallus gallus, Geobacillus,Gibberella zeae, Gossypium hirsutum, Homo sapiens, Kurtzmanomyces sp.,Leishmania infantum, Lycopersicon esculentum L, Malassezia furfur,Methanosarcina acetivorans, Mus musculus, Mus spretus, Mycobacteriumtuberculosis, Mycoplasma hyopneumoniae, Myxococcus xanthus, Neosartoryafischeri, Oryctolagus cuniculus, Oryza sativa, Penicillium cye/opium,Phlebotomus papatasi, Pseudomonas aeruginosa, Pseudomonas fluorescens,Pseudomonas Pseudomonas sp, Rattus norvegicus, Rhizomucor miehei,Rhizopus oryzae, Rhizopus stolonifer, Ricinus communis, Sarnia Cynthiaricini, Schizosaccharomyces pombe, Serratia marcescens, Spermophilustridecemlineatus, Staphylococcus simulans, Staphylococcus xylosus,Sulfolobus solfataricus, Sus scrofa, Thermomyces lanuginosus,Trichomonas vaginalis, Vibrio harveyi, Xenopus laevis, Yarrowialipolytica, a combination thereof, or a derivative thereof. It isappreciated that lipases derived from other organisms are similarlyoperable and are within the scope of the present invention.

A lipase is a “peptide,” “polypeptide,” and “protein” that are usedherein synonymously and are intended to mean a natural or syntheticcompound containing two or more amino acids having some level ofactivity toward a natural or synthetic substrate of a wild-type lipase.A wild-type lipase is a lipase that has an amino acid sequence identicalto that found in an organism in nature. An illustrative example of awild-type lipase is that found at GenBank Accession No. ACL68189 and SEQID NO: 1. An exemplary nucleotide sequence encoding a wild-type lipaseis found at Accession No. FJ536288. One of skill in the art recognizeshow to modify a nucleotide sequence to alter or create a proteinsequence.

Lipase activity is illustratively defined in units/gram. 1 unitillustratively corresponds to the amount of enzyme that hydrolyzes 1μmol acetic acid per minute at pH 7.4 and 40° C. using the substratetriacetin (Sigma-Aldrich, St. Louis, Mo., Product No. 90240). The lipaseof SEQ ID NO: 1 has an activity of approximately 200 units/gram.

Methods of screening for lipase activity are known and standard in theart. Illustratively, screening for lipase activity in a lipase proteinor analogue thereof illustratively includes contacting a lipase oranalogue thereof with a natural or synthetic substrate of a lipase andmeasuring the enzymatic cleavage of the substrate. Illustrativesubstrates for this purpose include tributyrin and triacetin both ofwhich are cleaved by a triacylglycerol lipase to liberate butyric acidor acetic acid respectively that is readily measured by techniques knownin the art.

Amino acids present in a lipase or analogue thereof illustrativelyinclude the common amino acids alanine, cysteine, aspartic acid,glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine,leucine, methionine, asparagine, proline, glutamine, arginine, serine,threonine, valine, tryptophan, and tyrosine; as well as less commonnaturally occurring amino acids, modified amino acids or syntheticcompounds, such as alpha-asparagine, 2-aminobutanoic acid or2-aminobutyric acid, 4-aminobutyric acid, 2-aminocapric acid(2-aminodecanoic acid), 6-aminocaproic acid, alpha-glutamine,2-aminoheptanoic acid, 6-aminohexanoic acid, alpha-aminoisobutyric acid(2-aminoalanine), 3-aminoisobutyric acid, beta-alanine,allo-hydroxylysine, allo-isoleucine, 4-amino-7-methylheptanoic acid,4-amino-5-phenylpentanoic acid, 2-aminopimelic acid,gamma-amino-beta-hydroxybenzenepentanoic acid, 2-aminosuberic acid,2-carboxyazetidine, beta-alanine, beta-aspartic acid, biphenylalanine,3,6-diaminohexanoic acid, butanoic acid, cyclobutyl alanine,cyclohexylalanine, cyclohexylglycine, N5-aminocarbonylomithine,cyclopentyl alanine, cyclopropyl alanine, 3-sulfoalanine,2,4-diaminobutanoic acid, diaminopropionic acid, 2,4-diaminobutyricacid, diphenyl alanine, N,N-dimethylglycine, diaminopimelic acid,2,3-diaminopropanoic acid, S-ethylthiocysteine, N-ethylasparagine,N-ethylglycine, 4-aza-phenylalanine, 4-fluoro-phenylalanine,gamma-glutamic acid, gamma-carboxyglutamic acid, hydroxyacetic acid,pyroglutamic acid, homoarginine, homocysteic acid, homocysteine,homohistidine, 2-hydroxyisovaleric acid, homophenylalanine, homoleucine,homoproline, homoserine, homoserine, 2-hydroxypentanoic acid,5-hydroxylysine, 4-hydroxyproline, 2-carboxyoctahydroindole,3-carboxylsoquinoline, isovaline, 2-hydroxypropanoic acid (lactic acid),mercaptoacetic acid, mercaptobutanoic acid, sarcosine,4-methyl-3-hydroxyproline, mercaptopropanoic acid, norleucine, nipecoticacid, nortyrosine, norvaline, omega-amino acid, ornithine, penicillamine(3-mercaptovaline), 2-phenylglycine, 2-carboxypiperidine, sarcosine(N-methylglycine), 2-amino-3-(4-sulfophenyl)propionic acid,1-amino-1-carboxycyclopentane, 3-thienylalanine,epsilon-N-trimethyllysine, 3-thiazolylalanine, thiazolidine 4-carboxylicacid, alpha-amino-2,4-dioxopyrimidinepropanoic acid, and2-naphthylalanine. A lipase includes peptides having between 2 and about1000 amino acids or having a molecular weight in the range of about150-350,000 Daltons.

A lipase is obtained by any of various methods known in the artillustratively including isolation from a cell or organism, chemicalsynthesis, expression of a nucleic acid sequence, and partial hydrolysisof proteins. Chemical methods of peptide synthesis are known in the artand include solid phase peptide synthesis and solution phase peptidesynthesis or by the method of Hackeng, T M, et al., Proc Natl Acad SciUSA, 1997; 94(15):7845-50, the contents of which are incorporated hereinby reference. A lipase included in an inventive composition may be anaturally occurring or non-naturally occurring protein. The term“naturally occurring” refers to a protein endogenous to a cell, tissueor organism and includes allelic variations. A non-naturally occurringpeptide is synthetic or produced apart from its naturally associatedorganism or is modified and is not found in an unmodified cell, tissue,or organism.

Modifications and changes can be made in the structure of a lipase andstill obtain a molecule having similar characteristics as lipase (e.g.,a conservative amino acid substitution). For example, certain aminoacids can be substituted for other amino acids in a sequence withoutappreciable loss of activity or optionally to reduce or increase theactivity of an unmodified lipase. Because it is the interactive capacityand nature of a polypeptide that defines that polypeptide's biologicalfunctional activity, certain amino acid sequence substitutions can bemade in a polypeptide sequence and nevertheless obtain a polypeptidewith like or other desired properties.

In making such changes, the hydropathic index of amino acids can beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a polypeptide is generallyunderstood in the art. It is known that certain amino acids can besubstituted for other amino acids having a similar hydropathic index orscore and still result in a polypeptide with similar biologicalactivity. Each amino acid has been assigned a hydropathic index on thebasis of its hydrophobicity and charge characteristics. Those indicesare: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine(+2.8); cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8);glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9);tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5);glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9);and arginine (−4.5).

It is believed that the relative hydropathic character of the amino aciddetermines the secondary structure of the resultant polypeptide, whichin turn defines the interaction of the polypeptide with other molecules,such as enzymes, substrates, receptors, antibodies, antigens, and thelike. It is known in the art that an amino acid can be substituted byanother amino acid having a similar hydropathic index and still obtain afunctionally equivalent polypeptide. In such changes, the substitutionusing amino acids whose hydropathic indices are within ±2, those within±1, and those within ±0.5 are optionally used.

Substitution of like amino acids can also be made on the basis ofhydrophilicity. The following hydrophilicity values have been assignedto amino acid residues: arginine (+3.0); lysine (+3.0); aspartate(+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2);glutamine (+0.2); glycine (0); proline (−0.5±1); threonine (−0.4);alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3);valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3);phenylalanine (−2.5); tryptophan (−3.4). It is understood that an aminoacid can be substituted for another having a similar hydrophilicityvalue and still obtain an enzymatically equivalent polypeptide. In suchchanges, the substitution of amino acids whose hydrophilicity values arewithin ±2, those within ±1, and those within ±0.5 are optionally used.

Amino acid substitutions are optionally based on the relative similarityof the amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and the like. Exemplarysubstitutions that take various of the foregoing characteristics intoconsideration are well known to those of skill in the art and include(original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys),(Asn: Gin, His), (Asp: Glu, Cys, Ser), (Gln: Asn), (Glu: Asp), (Gly:Ala), (His: Asn, Gln), (Ile: Leu, Val), (Leu: Ile, Val), (Lys: Arg),(Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe),and (Val: Ile, Leu). Embodiments of this disclosure, thus, contemplatefunctional or biological equivalents of a polypeptide as set forthabove. In particular, embodiments of the polypeptides can includeanalogues having about 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequenceidentity to a wild-type lipase.

It is further appreciated that the above characteristics are optionallytaken into account when producing a lipase with reduced or increasedenzymatic activity. Illustratively, substitutions in a substrate bindingsite, exosite, cofactor binding site, catalytic site, or other site in alipase protein may alter the activity of the enzyme toward a substrate.In considering such substitutions the sequences of other known naturallyoccurring or non-naturally occurring lipases may be taken into account.Illustratively, the N2915 substitution in lipoprotein lipase reduces itsenzymatic activity by 30-50 percent. Buskin, R. et al, FEBS Lett., 1995;367:257-262, the contents of which are incorporated herein by reference.Illustratively substitution at amino acid 95 in Rhizomucor miehei lipasecan produce a 2-3 fold increase in activity toward particularsubstrates. Similarly substitution at amino acid 94 can produce as muchas 6 times the activity of wild-type. Oh, S, et al, Biotech. Lett.,2001; 23: 563-568, the contents of which are incorporated herein byreference. Other substitutions at this or other sites may similarlyaffect enzymatic activity.

A lipase protein is illustratively recombinant. Methods of cloning,synthesizing or otherwise obtaining nucleic acid sequences encoding alipase are known and standard in the art that are equally applicable tolipase. Similarly, methods of cell transfection and protein expressionare similarly known in the art and are applicable herein. Such methodsare illustratively disclosed in Molecular Cloning: A Laboratory Manual,3rd ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 2001; Current Protocols in MolecularBiology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience,New York, 1992 (with periodic updates); and Short Protocols in MolecularBiology, ed. Ausubel et al., 52 ed., Wiley-Interscience, New York, 2002,the contents of each of which are incorporated herein by reference.

A lipase may be coexpressed with associated tags, modifications, otherproteins such as a fusion peptide, or other modifications orcombinations recognized in the art Illustrative tags include 6×His,FLAG, biotin, ubiquitin, SUMO, or other tag known in the art. A tag isillustratively cleavable such as by linking to lipase or an associatedprotein via an enzyme cleavage sequence that is cleavable by an enzymeknown in the art illustratively including Factor Xa, thrombin, SUMOstarprotein as obtainable from Lifesensors, Inc., Malvern, Pa., or trypsin.It is further appreciated that chemical cleavage is similarly operablewith an appropriate cleavable linker.

Protein expression is illustratively accomplished from transcription ofa lipase nucleic acid sequence, translation of RNA transcribed from thelipase nucleic acid sequence or analogues thereof. Protein expression isoptionally performed in a cell based system such as in E. coli, Helacells, or Chinese hamster ovary cells. It is appreciated that cell-freeexpression systems are similarly operable.

It is recognized that numerous analogues of lipase are operable andwithin the scope of the present invention including amino acidsubstitutions, alterations, modifications, or other amino acid changesthat increase, decrease, or do not alter the function of the lipaseprotein sequence. Several post-translational modifications are similarlyenvisioned as within the scope of the present invention illustrativelyincluding incorporation of a non-naturally occurring amino acid,phosphorylation, glycosylation, addition of pendent groups such asbiotinylation, fluorophores, lumiphores, radioactive groups, antigens,or other molecules.

An inventive method uses an inventive composition that is one or morelipases incorporated into a substrate itself or into a coating on thesubstrate. The lipase enzyme is optionally non-covalently associatedand/or covalently attached to the substrate or coating material or isotherwise associated therewith such as by bonding to the surface or byintermixing with the substrate/coating material during manufacture suchas to produce entrapped lipase. In some embodiments the lipase iscovalently attached to the substrate or coating material either bydirect covalent interaction between the lipase and one or morecomponents of the substrate or coating material or by association via alink moiety such as that described in U.S. Pat. App. Publ. No.2008/0119381, the contents of which are incorporated herein byreference.

There are several ways to associate lipase with a substrate or coating.One of which involves the application of covalent bonds. Specifically,free amine groups of the lipase may be covalently bound to an activegroup of the substrate. Such active groups include alcohol, thiol,aldehyde, carboxylic acid, anhydride, epoxy, ester, or any combinationthereof. This method of incorporating lipase delivers unique advantages.First, the covalent bonds tether the lipases permanently to thesubstrate and thus place them as an integral part of the finalcomposition with much less, if any at all, leakage of the lipase.Second, the covalent bonds provide extended enzyme lifetime. Over timeproteins typically lose activity because of the unfolding of theirpolypeptide chains. Chemical binding such as covalent bondingeffectively restricts such unfolding, and thus improves the proteinlife. The life of a protein is typically determined by comparing theamount of activity reduction of a protein that is free or beingphysically adsorbed with that of a protein covalently-immobilized over aperiod of time.

Lipases are optionally uniformly dispersed throughout the substratenetwork to create a substantially homogenous protein platform. In sodoing, lipases may be first modified with polymerizable groups. Themodified lipases may be solubilized into organic solvents in thepresence of surfactant, and thus engage the subsequent polymerizationwith monomers such as methyl methacrylate (MMA) or styrene in theorganic solution. The resultant composition includes lipase moleculeshomogeneously dispersed throughout the network.

Lipases are optionally attached to surfaces of a substrate. Anattachment of lipases corresponding to approximately 100% surfacecoverage was achieved with polystyrene particles with diameters rangefrom 100 to 1000 nm.

Chemical methods of lipase attachment to materials will naturally varydepending on the functional groups present in the lipase and in thematerial components. Many such methods exist. For example, methods ofattaching proteins (such as enzymes) to other substances are describedin O'Sullivan et al, Methods in Enzymology, 1981; 73:147-166 andErlanger, Methods in Enzymology, 1980; 70:85-104, each of which areherein incorporated herein by reference.

Lipases are optionally present in a coating that is layered upon asubstrate wherein the lipase is optionally entrapped in the coatingmaterial, admixed therewith, modified and integrated into the coatingmaterial or layered upon a coating similar to the mechanisms describedfor interactions between a lipase and substrate material.

Materials operable for interactions with a lipase to form an activesubstrate or coating illustratively include organic polymeric materials.The combination of these materials and a lipase form a protein-polymercomposite material that is used as a substrate material or a coating.

Methods of preparing protein-polymer composite materials illustrativelyinclude use of aqueous solutions of lipase and non-aqueous organicsolvent-borne polymers to produce bioactive organic solvent-borneprotein-polymer composite materials.

Methods of preparing protein-polymer composite materials areillustratively characterized by dispersion of lipase in solvent-borneresin prior to curing and in the composite materials. Lipases areoptionally dispersed in the protein-polymer composite material such thatthe lipases are unassociated with other bioactive proteins and/or formrelatively small particles of associated proteins. Illustratively, theaverage particle size of lipase particles in the protein-polymercomposite material is less than 10 μm (average diameter) such as in therange of 1 nm to 10 μm, inclusive.

Curable protein-polymer compositions are optionally two-componentsolvent-borne (2K SB) compositions. Optionally, one component systems(1K) are similarly operable. Illustratively, a lipase is entrapped in acoating material such as a latex or enamel paint, varnish, polyurethanegels, or other coating materials. Illustrative examples of incorporatingenzymes into paints are presented in U.S. Pat. No. 5,998,200, thecontents of which are incorporated herein by reference.

In two-component (2K) systems the two components are optionally mixedshortly before use, for instance, application of the curableprotein-polymer composition to a substrate to form a lipase containingcoating such as a bioactive clear coat. Generally described, the firstcomponent contains a crosslinkable polymer resin and the secondcomponent contains a crosslinker. Thus, the emulsion is a firstcomponent containing a crosslinkable resin and the crosslinker is asecond component, mixed together to produce the curable protein-polymercomposition.

A polymer resin included in methods and compositions of the presentinvention can be any film-forming polymer useful in coating or substratecompositions, illustratively clear coat compositions. Such polymersillustratively include, aminoplasts, melamine formaldehydes, carbamates,polyurethanes, polyacrylates, epoxies, polycarbonates, alkyds, vinyls,polyamides, polyolefins, phenolic resins, polyesters, polysiloxanes; andcombinations of any of these or other polymers.

In particular embodiments, a polymer resin is crosslinkable.Illustratively, a crosslinkable polymer has a functional groupcharacteristic of a crosslinkable polymer. Examples of such functionalgroups illustratively include acetoacetate, acid, amine, carboxyl,epoxy, hydroxyl, isocyanate, silane, vinyl, other operable functionalgroups, and combinations thereof.

Examples of organic crosslinkable polymer resins includes aminoplasts,melamine formaldehydes, carbamates, polyurethanes, polyacrylates,epoxies, polycarbonates, alkyds, vinyls, polyamides, polyolefins,phenolic resins, polyesters, polysiloxanes, or combinations thereof.

A crosslinking agent is optionally included in the composition. Theparticular crosslinker selected depends on the particular polymer resinused. Non-limiting examples of crosslinkers include compounds havingfunctional groups such as isocyanate functional groups, epoxy functionalgroups, aldehyde functional groups, and acid functionality.

In particular embodiments of protein-polyurethane composite materials, apolymer resin is a hydroxyl-functional acrylic polymer and thecrosslinker is a polyisocyanate.

A polyisocyanate, optionally a diisocyanate, is a crosslinker reactedwith the hydroxyl-functional acrylic polymer according to embodiments ofthe present invention. Aliphatic polyisocyanates are preferredpolyisocyanates used in processes for making protein-polymer compositematerials for clearcoat applications such as in automotive clearcoatapplications. Non-limiting examples of aliphatic polyisocyanates include1,4-butylene diisocyanate, 1,4-cyclohexane diisocyanate,1,2-diisocyanatopropane, 1,3-diisocyanatopropane, ethylene diisocyanate,lysine diisocyanate, 1,4-methylene bis(cyclohexyl isocyanate),diphenylmethane 4,4′-diisocyanate, an isocyanurate of diphenylmethane4,4′-diisocyanate, methylenebis-4,4′-isocyanatocyclohexane,1,6-hexamethylene diisocyanate, an isocyanurate of 1,6-hexamethylenediisocyanate, isophorone diisocyanate, an isocyanurate of isophoronediisocyanate, p-phenylene diisocyanate, toluene diisocyanate, anisocyanurate of toluene diisocyanate, triphenylmethane4,4′,4″-triisocyanate, tetramethyl xylene diisocyanate, and meta-xylenediisocyanate.

Curing modalities are those typically used for conventional curablepolymer compositions.

Lipase-polymer composite materials used in embodiments of the presentinvention are optionally thermoset protein-polymer composite materials.For example, a substrate or coating material is optionally cured bythermal curing. A thermal polymerization initiator is optionallyincluded in a curable composition. Thermal polymerization initiatorsillustratively include free radical initiators such as organic peroxidesand azo compounds. Examples of organic peroxide thermal initiatorsillustratively include benzoyl peroxide, dicumylperoxide, and laurylperoxide. An exemplary azo compound thermal initiator is2,2′-azobisisobutyronitrile.

Conventional curing temperatures and curing times can be used inprocesses according to embodiments of the present invention. Forexample, the curing time at specific temperatures, or under particularcuring conditions, is determined by the criteria that the cross-linkerfunctional groups are reduced to less than 5% of the total presentbefore curing. Cross-linker functional groups can be quantitativelycharacterized by FT-IR or other suitable method. For example, the curingtime at specific temperatures, or under particular curing conditions,for a polyurethane protein-polymer composite of the present inventioncan be determined by the criteria that the cross-linker functional groupNCO is reduced to less than 5% of the total present before curing. TheNCO group can be quantitatively characterized by FT-IR. Additionalmethods for assessing the extent of curing for particular resins arewell-known in the art. Illustratively, curing may include evaporation ofa solvent or by exposure to actinic radiation, such as ultraviolet,electron beam, microwave, visible, infrared, or gamma radiation.

One or more additives are optionally included for modifying theproperties of the lipase-polymer composite material and/or the admixtureof organic solvent and polymer resin, the aqueous lipase solution, theemulsion, and/or the curable composition. Illustrative examples of suchadditives include a UV absorbing agent, a plasticizer, a wetting agent,a preservative, a surfactant, a lubricant, a pigment, a filler, and anadditive such as an additive to increase sag resistance.

A substrate or coating including a lipase is illustratively an admixtureof a polymer resin, a surfactant and a non-aqueous organic solvent,mixed to produce an emulsion. The term “surfactant” refers to a surfaceactive agent that reduces the surface tension of a liquid in which it isdissolved, or that reduces interfacial tension between two liquids orbetween a liquid and a solid.

Surfactants used can be of any variety including amphoteric,silicone-based, fluorosurfactants, anionic, cationic and nonionic suchas described in K. R. Lange, Surfactants: A Practical Handbook, HanserGardner Publications, 1999; and R. M. Hill, Silicone Surfactants, CRCPress, 1999. Examples of anionic surfactants include alkyl sulfonates,alkylaryl sulfonates, alkyl sulfates, alkyl and alkylaryl disulfonates,sulfonated fatty acids, sulfates of hydroxyalkanols, sulfosuccinic acidesters, sulfates and sulfonates of polyethoxylated alkanols andalkylphenols. Examples of cationic surfactants include quaternarysurfactants and amineoxides. Examples of nonionic surfactants includealkoxylates, alkanolamides, fatty acid esters of sorbitol or manitol,and alkyl glucamides. Examples of silicone-based surfactants includesiloxane polyoxyalkylene copolymers.

When a surface which is optionally a substrate or a coated substrate, iscontacted with a fingerprint, the lipase enzyme or combinations ofenzymes contact the fingerprint, or components thereof. The contactingallows the enzymatic activity of the substrate or coating to interactwith and enzymatically alter the components of the fingerprint improvingtheir removal from the substrate or coating.

It is appreciated that the inventive methods of facilitating fingerprintremoval will function at any temperature whereby the lipase is active.Optionally, the inventive method is performed at 4° C. Optionally, aninventive method is performed at 25° C. Optionally, an inventive processis performed at ambient temperature. Some embodiments are performedbetween 40° C. and 120° C.

An inventive method optionally includes heating the substrate itself orapplying heat to the surface of the substrate. Heating is defined asincreasing the surface temperature of a substrate/coating to a levelhigher than the ambient temperature. In some embodiments heating israising the surface temperature by at least 5° C. Heating is optionallyraised by exposure to sunlight or other heat source. The surfacetemperature is optionally raised to such a level that the breakdownproducts volatilize to the point of no visual material remaining on thesubstrate within 24 hours. Optionally, the temperature is raised to sucha level that the breakdown products are removed to the point of novisual material remaining on the substrate within 0.5 to 3 hours,inclusive. Optionally, the substrate surface temperature is increased tobetween 40° C. and 125° C., inclusive.

Heat is optionally applied continuously or intermittently. Heat isoptionally applied until the breakdown products volatilize to the pointof no visual material remaining on the substrate. Optionally, heat isapplied for at least 30 minutes. In some embodiments, heat is appliedfor between 30 minutes to 6 hours, inclusive.

The presence of lipase combined with the material of a substrate or acoating on a substrate, optionally, with applied heat, breaks downfingerprint stains for facilitated fingerprint removal.

Various aspects of the present invention are illustrated by thefollowing non-limiting examples. The examples are for illustrativepurposes and are not a limitation on any practice of the presentinvention. It will be understood that variations and modifications canbe made without departing from the spirit and scope of the invention.

EXAMPLE 1

Production of lipase containing material operable for coating asubstrate.

Materials: Polyacrylate resin Desmophen A870 BA, and the hexamethylenediisocyanate (HDI) based polyfunctional aliphatic polyisocyanate resinDesmodur N 3600, the polyester resin Bayhydrol XP 7093, and Bayhydur 302are obtained from Bayer Corp. (Pittsburgh, Pa.). The surfactant BYK-333is obtained from BYK-Chemie (Wallingford, Conn.). α-Amylase KLEISTASESD80 from Bacillus subtilis (EC 3.2, 1.1), lipase PS from Burkholderiacepacia, and Lipase AP12 from Aspergillus niger are obtained from AmanoEnzyme Inc (Nagoya, Japan). Butyl acetate, Bradford reagent, bovineserum albumin (BSA) from bovine serum, starch from potatoes, starch fromwheat, maltose, sodium potassium tartrate, 3,5-dinitrosalicylic acid,Na₂(PO₄), NaCl, K₂(PO₄), casein, trichloroacetic acid, Folin &Ciocalteu's phenol reagent, Na₂(CO₃), sodium acetate, calcium acetate,tyrosine, p-nitrophenyl palmitate, ethanol, iodine, glucose, maltose,maltotriose, maltohexose, dextrin with different molecular weight (10kDa, 40 kDa) were obtained from Sigma Chemical Co., St. Louis, Mo.,U.S.A. Aluminum panels and 8-path wet film applicators are obtained fromPaul N. Gardner Company, Inc. (Pompano Beach, Fla.).

Enzyme preparation: Lipase AP12 is subjected to purification from a 150mL solution containing 7.5 g crude lipase powder in DI water at aprotein concentration of about 5 mg/mL (determined by the Bradfordmethod). All solutions are kept on ice. Ultrafiltration is performedusing a 150 mL Amicon cell (Millipore, Billerica, Mass.) at a pressureof 55 psi using a 30 kDa cut-off membrane. Ultrafiltration is repeated 3times by adding DI water to the remaining concentrated solution to avolume of 150 mL after each run. The final remaining purified proteinsolution is used to prepare coatings typically at a lipase concentrationof 200 mg/mL.

Incorporation of lipase in to form protein-polymer coating:Solvent-borne two-component polyurethane (PU) is formed by thepolymerization of Desmophen A 870 BA and Desmodur N 3600 at a weightratio of 2.6:1 of Desmophen A 870 BA to Desmodur N 3600. Typically, 2.1g of Desmophen A 870 BA (solid content of 70 wt %), 0.0167 g of BYK-333dissolved in 100 μl of n-butanol, and 0.5 mL of butyl acetate are mixedin a 20 mL vial. 600 μl of 200 mg/mL purified lipase enzyme solution isadded and mixed for 1 minute to form a white emulsion. Subsequently, 0.8g of Desmodur N 3600 is added and mixed manually for 1 minute. Theresulting curable protein-polymer solution is then applied to aluminumtesting panels via drawdown using an 8-path applicator. The resultingcoating is cured at 80° C. for 24 hours.

EXAMPLE 2

Fingerprint removal: The lipase containing coated panels of Example 1are loaded with human fingerprints after touching the skin of the faceor forearms. Fingerprinted panels are incubated at room temperature forat least 24 hours. A control panel is coated with the coating of Example1 that is free of enzyme. After this first incubation period, the coatedsubstrate is incubated in an oven at a temperature of 65° C. or higherfor 1 to 6 hours.

FIG. 1 demonstrates that incubation of the enzyme coated panels at 65°C. for two hours facilitates complete removal of fingerprints. (B:control; L: lipase; LA: combined lipase and amylase in coating.)

EXAMPLE 3

The rate of fingerprint removal is faster in an enzyme containingsubstrate than control. FIG. 2 demonstrates aluminum plates coated withcontrol or enzyme containing protein-polymer coatings as described inExample 1 where a fingerprint is loaded at the interface between the twocoatings as described in Example 2. FIG. 2A demonstrates the remainingfingerprint after incubation at ambient temperature for 3 days. Theplates are subsequently incubated for 2.5 hours at 65° C. andphotographs are taken at various intervals as illustrated in FIG. 2B.FIG. 3 demonstrates that the increased rate of fingerprint removal isindependent of the source of the fingerprints.

Various modifications of the present invention, in addition to thoseshown and described herein, will be apparent to those skilled in the artof the above description. Such modifications are also intended to fallwithin the scope of the appended claims.

It is appreciated that all reagents are obtainable by sources known inthe art unless otherwise specified. Methods of nucleotide amplification,cell transfection, and protein expression and purification are similarlywithin the level of skill in the art.

Patents and publications mentioned in the specification are indicativeof the levels of those skilled in the art to which the inventionpertains. These patents and publications are incorporated herein byreference to the same extent as if each individual application orpublication was specifically and individually incorporated herein byreference.

The foregoing description is illustrative of particular embodiments ofthe invention, but is not meant to be a limitation upon the practicethereof. The following claims, including all equivalents thereof, areintended to define the scope of the invention.

1. A method of facilitating the removal of a fingerprint on a substrateor a coating comprising: providing a substrate or a coating; associatinga lipase with said substrate or said coating such that said lipase iscapable of enzymatically degrading a component of a fingerprint, andfacilitating the removal of a fingerprint by vaporization from thelipase associated substrate or coating when contacted by a fingerprint.2. The method of claim 1 wherein said lipase is covalently attached tosaid substrate or to said coating.
 3. The method of claim 1 wherein saidlipase is non-covalently adhered to or admixed into said substrate orsaid coating.
 4. The method of claim 1 comprising heating said substrateor said coating or applying heat to a surface of said substrate or saidcoating subsequent to being contacted by a fingerprint.
 5. The method ofclaim 4 wherein said heating is for at least 30 minutes.
 6. The methodof claim 1 wherein said substrate or said coating comprises an organiccrosslinkable polymer resin.
 7. The method of claim 6 wherein saidorganic crosslinkable polymer resin comprises a functional group ofacetoacetate, acid, amine, carboxyl, epoxy, hydroxyl, isocyanate,silane, vinyl, or combinations thereof.
 8. The method of claim 6 whereinsaid organic crosslinkable polymer resin is aminoplasts, melamineformaldehydes, carbamates, polyurethanes, polyacrylates, epoxies,polycarbonates, alkyds, vinyls, polyamides, polyolefins, phenolicresins, polyesters, polysiloxanes, or combinations thereof.
 9. Themethod of claim 6 wherein said organic crosslinkable polymer is ahydroxyl-functionalized acrylate resin.
 10. The method of claim 1wherein said lipase is lipoprotein lipase, acylglycerol lipase,hormone-sensitive lipase, phospholipase A1, phospholipase A2,phospholipase C, phospholipase D, phosphoinositide phospholipase C, alysophospholipase, or a galactolipase.
 11. The method of claim 1 whereinsaid lipase is a triacylglycerol lipase.